京都大学 / 新制・課程博士 / 博士(理学) / 甲第23740号 / 理博第4830号 / 新制||理||1691(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 教授 松下 智直, 准教授 竹中 瑞樹 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The lower oxygen limit (LOL) in plants marks the oxygen (O2) level where the metabolism shifts from being predominantly aerobic to anaerobic; recent work has shown that respiratory-based indicators of this metabolic shift are well-correlated with changes in chlorophyll a fluorescence signals. The physiological and biochemical changes at the root of this relationship have not been well-described in the literature. The processes involved are spatially separated: chlorophyll fluorescence is associated with the lightdependent reactions and emanates from the chloroplasts whereas aerobic respiration and fermentation occurs in the mitochondria and cytosol, respectively. Evidences outlined in this thesis are used to suggest the mechanistic link between these three regions of the cell is a fluid exchange of cellular reductant. When mitochondrial respiration is inhibited as a result of inadequate O2, used as a terminal electron acceptor, glycolytic reductant in the form of NADH accumulates in the cytosol. Reductant imbalances between the cytosol and organelles can be adjusted indirectly using translocators. Excess chloroplastic reductant is used to reduce the plastoquinone (PQ) pool via NADPH-dehydrogenase, a component of the chlororespiratory pathway, effectively decreasing the photochemical quenching (qP) capacity thereby inducing a switch from minimum fluorescence (Fo) to a higher relative fluorescence (F) value where qP < 1. Subjecting dark-adapted photosystems to low-intensity light increased Fo to a slightly higher F value due to a lightinduced reduction of the oxidized PQ pool when the O2 was above the LOL, but decreased F as a result of a PSI-driven oxidation of the already over-reduced PQ pool when the O2 was below the LOL. Low O2 was also shown to increase violaxanthin deepoxidation and non-photochemical quenching (qN), likely a reflection of the overreduced state of the photosystems and associated pH decrease. Dynamic controlled atmosphere (DCA) is a fluorescence-based controlled atmosphere (CA) system that sets the optimum atmosphere for fruits and vegetables based on a product’s fluorescence response. Experiments in this thesis on the relationship between O2, temperature, light, metabolism, pigmentation and chlorophyll fluorescence were used to interpret the physiology behind fluorescence changes, suggest improved DCA techniques and outline potentially profitable avenues for future research.

Plants absorb light for photosynthesis, but not all is used. Excess light energy may lead to photoinhibition of photosynthesis and irreversible photooxidative damage. Plants have evolved mechanisms for energy dissipation under high light stress. One such response may involve production of ELIP. It is of interest to know what signal(s) may be involved in ELIP expression. My hypothesis is that redox status of the chloroplast photosynthetic electron transport Chain (PETC) and/or chlororespiration may induce ELIP expression. Using the Arabidopsis thaliana immutans (im) chlororespiratory mutant, this hypothesis was tested. Etiolated seedlings of this variegated mutant were subjected to various light intensities over 0-24 hr period and ELIP mRNA levels were analyzed. These were compared with the wild type plants treated in the same manner. It was found that mature thylakoids may not be required for ELIP expression, and that both photoreceptor-dependent and independent components may be involved in ELIP expression.

Le mutant immutans d'Arabidopsis présente un phénotype panaché et accumule du phytoène, précurseur des caroténoïdes.<br />Le gène IMMUTANS code une protéine plastidiale, homologue à l'oxydase alterne mitochondriale, et loacalisée dans les lamelles stromatiques du thylacoïde et dans les membranes achlorophylliques des chromoplastes. Son expression chez E. coli permet l'établissement d'un transport d'électrons résisatn au KCN et inhibé par le n-PG.<br />Cette protéine apparait être une oxydase terminale plastidiale (PTOX), dont l'activité dépend de la présence de PQ et de fer.<br />PTOX semblerait églaement être l'oxydase terminale de la chlororespiration; exprimée chez le tabac, elle accélère la ré-oxydation non photochimique des PQ, et entraine un flux d'électrons vers l'O2, évitant ainsi la sur-réduction des PQ. La forte expression de PTOX chez le plante d'altitude Ranunculus glacialis pourrait indiquer un role particulièrement actif de PTOX dans les mécanismes de résistance à la photoinhibition.

Chez les plantes supérieures, les membranes thylacoïdiennes des chloroplastes contiennent des protéines de type respiratoire, un complexe NADH déshydrogénase (Ndh) homologue au complexe I bactérien et une oxydase (PTOX) homologue à l'oxydase alternative des mitochondries. Dans le but d'élucider la fonction du complexe Ndh, le gène chloroplastique ndhB a été inactivé par transformation chloroplastique du tabac. Chez les plantes transplastomiques obtenues, des mesures de fluorescence de la chlorophylle ont permis de démontrer que ce complexe est impliqué dans la réduction non photochimique du pool de plastoquinones (PQ). Chez les mutants dépourvus de complexe Ndh, un retard de croissance ainsi qu'une altération des capacités photosynthétiques ont été observés dans des conditions où la photorespiration est stimulée par une limitation de la disponibilité en CO2 (par exemple un déficit hydrique modéré). En étudiant les effets de l'antimycine A, un inhibiteur du transfert cyclique des électrons autour du PS I, sur l'activité photosynthétique de ces plantes, nous avons conclu que le complexe Ndh est impliqué dans une voie de transfert cyclique et participe à la fourniture en ATP nécessaire à la fixation de CO2 quand la demande est forte. <br />Afin de déterminer le rôle de PTOX, nous avons généré des plantes de tabac surexprimant cette protéine. Des mesures de fluorescence de la chlorophylle nous ont permis de démontrer l'implication de PTOX dans l'oxydation non photochimique du pool de PQ. Nous avons conclu que le complexe Ndh et PTOX, tous deux localisés dans les thylacoïdes lamellaires, constituent avec le pool de plastoquinones les éléments d'une chaîne de transfert d'électrons de type chlororespiratoire. Nous avons proposé un mécanisme par lequel l'activité du transfert cyclique des électrons autour du PS I serait régulée par l'activité de la chlororespiration, à travers un contrôle fin du niveau rédox de certains transporteurs de la chaîne de transfert d'électrons.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cloroplasto à uma organela caracterÃstica dos organismos fotossintetizantes sendo seu papel primordial na geraÃÃo de energia a partir de gÃs carbÃnico e Ãgua. Essa organela pode ter seu funcionamento comprometido quando submetida a estresses ambientais devido a fragilidade e complexidade do sistema. Para evitar perdas provocadas pelo estresse existem vÃrios mecanismos de adaptaÃÃo e regulaÃÃo das reaÃÃes que ocorrem no cloroplasto. Recentemente caracterizou-se mais um desses provÃveis mecanismos que foi chamado de clororespiraÃÃo. A clororespiraÃÃo foi esclarecida com a descoberta de uma enzima similar a oxidase alternativa da mitocondria que chamou-se de oxidase terminal do plastÃdeo (PTOX). A funÃÃo dessa respiraÃÃo do cloroplasto permanece incerta, mas uma das hipÃteses mais aceitas à que o funcionamento da clororespiraÃÃo poderia prevenir a formaÃÃo de espÃcies reativas de oxigÃnio atravÃs da reciclagem dos intermediÃrios redutores do cloroplasto. No presente trabalho foi caracterizado a presenÃa de dois genes que codificam para a oxidase terminal do plastÃdeo em plantas de Zea mays. Estudou-se tambÃm a expressÃo diferencial de ambos genes da PTOX em resposta ou estresse hÃdrico, alÃm da caracterizaÃÃo da clororespiraÃÃo atravÃs da atividade da NADH desidrogenase plastidial (NDH) em gel de poliacrilamida. A caracterizaÃÃo molecular dos genes da PTOX mostrou homologia de 60% quando comparadas as sequÃncias dos genes e de 79% quando comparadas as prÃ-proteÃnas traduzidas. Os genes dessa proteÃna tÃm estruturas similares, sendo compostos por oito introns e 9 Ãxons. Um estudo das regiÃes dos promotores dos genes mostrou que existiam elementos comuns porÃm a presenÃa de elementos diferentes como, o elementos cis MBS que à responssivo à seca, poderia revelar uma regulaÃÃo diferencial dos genes. A resposta diferencial foi confirmada atravÃs de RT-PCR semiquantitativo. O gene chamado de ptox1 teve sua expressÃo estÃvel, podendo ser considerado um gene constitutivo, enquanto que o gene chamado de ptox2 teve um aumento da expressÃo proporcional ao estresse aplicado tanto em folhas como em raÃzes de plantas de milho. A anÃlise da atividade da NDH em gel (zimograma) revelou a presenÃa dessa enzima em cloroplastos de milho confirmando a presenÃa das enzimas da clororespiraÃÃo. O estudo filogenÃtico de sequencias de cDNA de bancos de dados mostraram que milho e sorgo pertencentes ao grupo das monocotiledÃneas, sÃo espÃcies muito prÃximas e que compartilham dois genes ortÃlogos da PTOX identificados como ptox1 e ptox2. Concluiu-se pela primeira vez a presenÃa de dois genes da PTOX no genoma do milho, uma monocotiledÃena de metabolismo C4. Os genes foram denominados de ptox1 e ptox2. Eles foram encontrados em raÃzes e folhas e apenas o gene da ptox2 pareceu ser induzido em resposta ao estresse osmÃtico. / The chloroplast is an organelle characteristic of photosynthetic organisms and their role in generating energy from carbon dioxide and water. This organelle may be functionally compromised when subjected to environmental stress due to the fragility and complexity of the system. To avoid losses caused by stresses plants have evolved various coping mechanisms, as well as, regulation of the reactions that occur in the chloroplast. Most recently it was characterized one of these mechanisms that was called chlororespiration. The chlororespiration was bring to light with the discovery of an enzyme, similar to the alternative oxidase of mitochondria, that was called the plastid terminal oxidase (PTOX). The function of this chloroplast respiration remains uncertain, but one of the most accepted hypothesis is that the operation of chlororespiration could prevent the formation of reactive oxygen species by recycling the reducing intermediates of the chloroplast. The present study characterized the presence of two genes encoding the plastid terminal oxidase in plants of Zea mays., and its differential expression in response to water stress. It was also characterized the chlororespiration through the activity of plastidial NADH dehydrogenase (NDH) in polyacrylamide gel. The molecular characterization of PTOX genes showed 60% homology when compared sequences of genes, but 79% when compared to pretranslated proteins. The genes of this protein have similar structures, being composed of nine exons and eight introns. A study of regions of the promoters of the genes showed that there were common elements, but the presence of different elements such as the cis elements that MBS responsive to drought, could reveal a differential regulation of genes. The differential response was confirmed by semiquantitative RT-PCR. The gene called ptox1 had its expression level stable and could be considered a constitutive gene, while the gene called ptox2 had an increased expression proportional to the applied stress in both leaves and roots of maize plants. The analysis of NDH activity gel (zimograms) revealed the presence of this enzyme in maize chloroplasts suggesting the existence of the chlororespiratory pathway. The phylogenetic analysis of cDNA sequences from NCBI databases showed that maize and sorghum, being closely related species, share two genes )identified as orthologs of PTOX (ptox1 and ptox2). It was confirmed for the first time the presence of two PTOX genes in the genome of maize, a C4-metabolism monocotyledon and its differential expression under drought stress.